The long-term goal of this proposal is to advance the physiological and potentially therapeutic understanding of angiotensin type 2 receptors (AT2R) in kidney function and blood pressure regulation, particularly in obesity. During the previous funding period, we clearly demonstrated that renal AT2R are transcriptionally up-regulated in obese rats. The activation of these receptors promotes natriuresis mainly via proximal tubule mechanism, and protects against blood pressure increase, especially in high Na-fed obese rats. Interestingly, however, we observed that AT2R has positive functional interaction with Ang(1-7)/MasR, which is considered as protective arm of the renin angiotensin system (RAS). Specifically, we for the first time observed that pharmacological activation of AT2R in obese rats by specific agonists caused a marked increase in the kidney levels of Ang(1-7), a peptide hormone known to promote natriuresis by acting on MasR. This positive regulatory association between AT2R and Ang(1-7) was further supported by AT2R knockout mice. Further, the activity of neprilysin (NEP), a peptidase which generates Ang(1-7) from Ang I, was proportionally increased in the kidney cortex of AT2R agonist-treated obese rats. Moreover, NEP activity is negatively regulated by phosphorylation at Ser6 of NEP by the MEK/ERK1/2 pathway, which is potentially inhibited by AT2R. Preliminary studies revealed that AT2R-mediated natriuresis in obese rats was dependent on MasR activation and vice-versa, suggesting a functional inter-dependence of these receptors. This notion is further supported by additional preliminary studies on co-immunoprecipitation and FRET between AT2R and MasR. Collectively, we hypothesize that the AT2R and MasR create a functional dimer to mediate cell signaling and promote natriuresis. This natriuretic function is intensified by an AT2R-mediated increase in Ang(1-7) levels generated by increased NEP activity. The increase in NEP results from inhibition of MEK/ERK1/2 by the AT2R. Two specific aims are developed. Aim 1 will determine that AT2R and MasR functionally interact to stimulate cell signaling and promote natriuresis. Aim 2 will determine that AT2R increases Ang(1-7) levels by inhibiting MAP kinase pathway, thereby enhancing NEP activity. We will employ physiological, biochemical and molecular approach for accomplishing these aims. Studies in kidney specific knockdown (KD) animals (AT2R KD, MasR KD, ACE2 KD, NEP KD) will be complemented by pharmacological manipulation of these target molecules. The proposed studies will significantly impact our understanding of the increasingly complex regulation of renal physiology by RAS. Specifically, our studies will lead to a greater appreciation of the role the Ang(1-7)/NEP(ACE2)/MasR axis plays in modulating natriuretic response to AT2R activation. We anticipate that this understanding will reveal AT2R, by virtue of its positive regulation of the protective arm of RAS, as a novel therapeutic target to reverse kidney dysfunction and treat hypertension, especially in obesity.